NAPRT Expression and Epigenetic Regulation in Pediatric Rhabdomyosarcoma as a Potential Biomarker for NAMPT Inhibition.

New treatments are needed to improve survival in children with rhabdomyosarcoma (RMS). Nicotinamide adenine dinucleotide (NAD+) biosynthesis, regulated by the enzymes nicotinic acid phosphoribosyltransferase (NAPRT) and nicotinamide phosphoribosyltransferase (NAMPT), represents a metabolic vulnerability due to high NAD+ turnover in cancers. Although NAMPT inhibitors (NAMPTi) show preclinical promise, clinical translation has been limited by toxicity and the lack of predictive biomarkers. In this study, we evaluated NAPRT expression in RMS and its potential as an actionable biomarker to guide NAMPTi therapy. NAPRT promoter methylation, transcript levels, and protein expression were assessed in RMS cells, patient-derived xenografts (PDX), and primary tumors (n = 109) from the Children's Oncology Group. In vitro sensitivity to NAMPTi was tested in isogenic RMS cell lines, examining the role of NAPRT expression in mediating cytotoxicity and the ability of nicotinic acid (NA) to rescue viability. In vivo efficacy was assessed using cell-derived, NAPRT-isogenic, and orthotopic PDX models. NAPRT promoter hypermethylation was found in a subset of RMS models and patient samples. Immunohistochemistry showed loss of NAPRT protein in 30% to 40% of tumors, defined as <1% tumor cell staining. Methylation modestly correlated with protein expression. NAPRT-deficient cells were highly sensitive to NAMPTi, driven by NAD+ depletion and not reversible with NA. In vivo, NAMPTi induced significant tumor regression, which was not abrogated with NA administration in NAPRT-deficient models. NAPRT loss occurs in a subset of RMS and may offer a biomarker-driven strategy to expand the therapeutic window of NAMPTi. Further research is needed to understand NAPRT regulation and optimize biomarker assays in future clinical trials.